Metabolomics-Based Analysis of Miniature Flask Contents Identifies
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www.nature.com/scientificreports OPEN Metabolomics‑based analysis of miniature fask contents identifes tobacco mixture use among the ancient Maya Mario Zimmermann1*, Korey J. Brownstein4,5, Luis Pantoja Díaz2, Iliana Ancona Aragón2, Scott Hutson3, Barry Kidder3, Shannon Tushingham1 & David R. Gang4 A particular type of miniature ceramic vessel locally known as “veneneras” is occasionally found during archaeological excavations in the Maya Area. To date, only one study of a collection of such containers successfully identifed organic residues through coupled chromatography–mass spectrometry methods. That study identifed traces of nicotine likely associated with tobacco. Here we present a more complete picture by analyzing a suite of possible complementary ingredients in tobacco mixtures across a collection of 14 miniature vessels. The collection includes four diferent vessel forms and allows for the comparison of specimens which had previously formed part of museum exhibitions with recently excavated, untreated containers. Archaeological samples were compared with fresh as well as cured reference materials from two diferent species of tobacco (Nicotiana tabacum and N. rustica). In addition, we sampled six more plants which are linked to mind‑altering practices through Mesoamerican ethnohistoric or ethnographic records. Analyses were conducted using UPLC‑MS metabolomics‑based analytical techniques, which signifcantly expand the possible detection of chemical compounds compared to previous biomarker‑focused studies. Results include the detection of more than 9000 residual chemical features. We trace, for the frst time, the presence of Mexican marigold (Tagetes lucida) in presumptive polydrug mixtures. Te induction of altered states of consciousness (ASC) is a common feature of humankind1, among hunting and gathering communities2 as well as complex societies3,4. ASCs cause either a heightened awareness of the senses or the perception of alternate realities. How these states are achieved varies profusely between cultures, yet there are two principal pathways: the creation or activation of pathomorphic stressors (through, e.g., fast- ing, sleep deprivation, bloodletting), which lead to the liberation of endogenous opioid peptides (endorphins)5, and the administration of entheogenic substances of either plant or animal origin6. Either way, many societies have specialists, such as shamans, traditional healers, and doctors, who help community members in achieving ASC7. Moreover, McClenon 8 argues the susceptibility to shamanic/hypnotic suggestion was positively selected in humans and their ancestors for the evolutionary benefts of pain reduction, enhanced healing, and easier childbirth. Among entheogenic techniques, smoking in pipes is the most likely to be recognized archaeologically. How- ever, although Pleistocene hunter-gatherers settling the Americas from Siberia arguably brought along a complex of shamanistic practices and medicinal plant use 9, “nicotine delivery devices” (NDD) are an innovation not appearing in the North American archaeological record before the Late Archaic. Te earliest known dates for the US southwest and northeast fall within the frst millennium BCE10, while to this day the oldest fnds from the Columbia River Basin reach back only 1200 years11. Tobacco (Nicotiana sp.) is the most prolifc producer of nicotine12, but this metabolite has been detected in lower concentrations in other genera13,14. Nicotiana species also contain the psychoactive harmala alkaloids harman and norharman, which are known for their efects in 1Department of Anthropology, Washington State University, P.O. Box 644910, Pullman, WA 99164-4910, USA. 2Centro Regional Yucatán, National Institute of Anthropology and History, Col. Gonzalo Guerrero, Calle 10 #310-A, 97310 Mérida, Yucatán, Mexico. 3Department of Anthropology, University of Kentucky, 211 Laferty Hall, Lexington, KY 40506-0027, USA. 4Institute of Biological Chemistry, Washington State University, P.O. Box 646340, Pullman, WA 99164-6340, USA. 5Present address: Department of Molecular Genetics and Cell Biology, The University of Chicago, Cummings Life Science Center, 920 E. 58th Street, Suite 1106, Chicago, IL 60637, USA. *email: [email protected] Scientifc Reports | (2021) 11:1590 | https://doi.org/10.1038/s41598-021-81158-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ drugs like ayahuasca15. Even though tobacco was widely documented as both a medicine and a sacred plant among Native Americans16,17, it was merely part of a much larger complex of psychoactive products, including plants that were smoked and that single-handedly account for dozens of genera 18. Terefore, archaeological research of the co-evolution of humans and drugs requires thorough methodological foundations and state-of- the-art technological tools. In this paper, we present a metabolomics-based case study from Mesoamerica with a focus on tobacco preparations as a window into the modes of drug consumption. In Mesoamerica, native communities such as the Nahua and Maya feature ritual specialists or shamans who communicate with supernatural entities and ancestors through ASCs induced by ingesting psychoactive plants as well as other pathways. De la Garza19 states tobacco is likely the most important sacred plant for the ritual and daily lives of Mesoamerican people. Interestingly, she specifcally refers to N. rustica rather than the more commonly known N. tabacum, which forms the base of present-day commercial products. With access being socially restricted, ASC-inducing plant products were most likely kept in specialized containers in the past. De la Garza19 argues the venenera fasks of the Maya Area are a strong candidate for such artifacts. Although the vernacular term venenera translates to “venom bottle”, these vessels have also been said to contain poison, medicine, pigment, or snuf. Troughout this article, we follow Loughmiller-Cardinal and Zagorevski20 in not making a priori assumptions about vessel contents and apply the neutral term “miniature fasks”. While information on storage containers is scarce, ethnohistoric sources from Mesoamerica provide more background on delivery devices and the mind-altering products themselves. Friar Bernardino de Sahagun was the frst outside observer to give detail on indigenous drug consumption in what is today central Mexico. He describes smoking products among contact-period Nahua in the following words: Tere are many ways of these canes, and they are made from many and diverse fragrant herbs, ground and mixed with each other, and flled and packed of roses, of fragrant spices, of the bitumen known as chapuputli, and of mushrooms, and of roses called poyomalli, or of tlzyetl, which is an herb. Sahagún 1999 [1569]:574. Based on ethnographic observation 21, Mexican marigold (Tagetes lucida) fgures notably among these tobacco mixture ingredients. In Mexico and Guatemala, this annual herb is commonly known for its role in ceremonies for the dead, which appear to have pre-Columbian roots22. However, present-day Huichol communities in west- ern Mexico continue to smoke dried leaves and fowers either by themselves or in a mixture with N. rustica21. Similar to most of their Mesoamerican neighbors, the ancient Maya did not use stone or clay pipes but other, more degradable NDDs such as reeds and corn husks 23. However, smoking was not the only way tobacco was consumed by the indigenous groups of Mesoamerica. Goodman24 states chewing, drinking, snufng, and enemas were all common forms of tobacco ingestion in the pre-Contact Americas. A contemporary of Sahagún’s Historia General de las Cosas de Nueva España, the Descripción de San Bartolomé (1588), for example, mentions chewing of pulverized tobacco mixed with lime by the Tz’utujil Maya of highland Guatemala. Te preparation was recog- nized for its strengthening and thirst-quenching properties and kept in small gourds 25. More recent ethnographic observations demonstrate the persistence of these consumption and storage patterns for tobacco-based products. Winter26 reports Mazatec of Oaxaca consistently carrying powdered green tobacco leaves in little gourds. Te powder is used to relieve fatigue and in witchcraf. Similar practices are observed for the Huichol27 and the Tzotzil Maya of Chiapas who call the powder-lime mixture mai28, a term used for tobacco by several Maya sub-groups. In an efort to trace tobacco consumption among the pre-Columbian Maya, Zagorevski and Loughmiller- Newman29 successfully identifed nicotine residues in a ceramic miniature vessel using liquid chromatogra- phy–mass spectrometry (LC–MS) methods. Although there is no provenience information for the fask, the ceramic type situates it in the Southern Maya Lowlands and the Late Classic (AD 550-850) period. Te container is of particular interest, because of a series of painted hieroglyphs reading yotoot may—the home of tobacco 30. Te authors point out that the lack of derivatives like cotinine, as well as the seemingly natural levels of nicotine enantiomer ratios, make the presence of thermo-altered tobacco unlikely. In other words, the vessel appears to have held a “fresh” tobacco product rather than its consumed remains29. Based on the available ethnohistoric and ethnographic data from larger Mesoamerica, our working hypothesis states that some of the miniature fasks found in the Maya Area contained mixtures of lime (likely Ca[OH]2), tobacco (Nicotiana sp.), and other psychoactive and/or aromatic plants. Te creation of an alkaline oral environ-